1. Field of the Invention
The present invention relates to an biological substance bound object given a label for identification for discriminating the type of each of a plurality of objects to which biological substances are individually bound, specifically the type of each of these biological substances, and a method for producing the same.
The present invention further relates to a method for discriminating a probe possessed by the object, a method for detecting, identifying and quantitatively measuring a probe bound to the object, and a method for detecting, identifying and quantitatively measuring a target substance bound to the probe.
Further, the present invention relates to a nucleic acid primer bound object given a label for identification for discriminating the type of each of a plurality of objects to which a plurality of types of nucleic acid primers are individually bound, specifically the type of each of the nucleic acid primers bound on the objects, and a method for producing the same, and a method for discriminating the type of the object, specifically the type of nucleic acid primer bound on the object. Further, the present invention relates to a method for using such a nucleic acid primer bound object given a label for identification to identify and detect or quantitatively measure the type of nucleic acid primer bound to the object, or a method for using a nucleic acid primer bound to the object to detect a single nucleotide polymorphism of a target nucleic acid.
2. Description of the Related Art
When a plurality of types of objects are treated, it is required to accurately discriminate the type of each of the objects in many cases. Particularly, if a plurality of types of objects to be treated cannot easily be discriminated by appearances, a label for identification corresponding to the type of each of objects is given to help discriminate the type of each of the objects.
For labeling for identification, there are various kinds of methods, but in essence, discriminable coded identifiers (codes), the number of which is equal to or greater than the number of types, to be discriminated, are assigned to individual types. If the object size of a target to which a label is give is large, character string type identifiers are used, in many cases, as discriminable coded identifiers (codes) used. For example, barcodes correspond to identifiers which are matched with character string type identifiers and converted into information of line widths and intervals of bars in accordance with a predetermined rule. Fundamentally, many of character string type identifiers employ a form in which a plurality of hierarchical label portions such as, for example, country codes (country numbers), area codes (toll numbers) and individual numbers in an area (in-area exchange numbers+individual line numbers) in the telephone number, are combined and integrated into one character string type identifier in order to more conveniently assign identifiers that can be expressed by a series of serial numbers.
If the object size of a target to which a label is given is small, it is technically difficult to give a visually discriminable character string type identifier described above. For example, an IC tag employs a method in which as a character string type identifier, information electronically described on an IC chip is identified via a digitized electromagnetic wave between the IC tag and an external detector. The size of this IC tag itself is in the order of 100 μm, and giving a character string type identifier to an object size of a dozen or so μm or less, and several μm or less in some cases is more technically difficult challenge.
For instance, one example of the case where the object size is a dozen or so μm or less and several μm or less in some cases is an object having probe molecules fixed on the surfaces of microscopic particles, which is used for detection of an biological substance.
For example, methods of examining a taxonomic near relative relationship by evaluating cross reactivity of antibody molecules specific to analogous microorganisms to an antigen on the surface of a target microorganism when classifying various kinds of microorganisms have been used since long ago. At this time, antibody molecules for use as a probe are fixed on the surfaces of gold fine particles, and integration of gold fine particles on the periphery of microorganisms are observed to determine presence/absence of a cross reaction. As a method for identifying an epitope sequence identified by various kinds of antibody molecules, a method in which for example, colibacillus strains expressing peptides undergoing an antigen/antibody reaction with antibody molecules fixed on the surfaces of microscopic ferrite particles are separated from random peptide library displayed by colibacillus bacteria by applying a panning method is widely known. In this method, only colibacillus strains expressing peptides having amino acid sequences capable of reacting with antibody molecules are separated in the form of being coupled to ferrite particles when magnetically microscopic ferrite particles are collected.
A main advantage possessed by microscopic particles fixing probe molecules, which are used for detection of an biological substance, is that by using means for separating microscopic particles as a solid matter from a liquid phase, a detection target substance bound by probe molecules and other contaminants existing in the liquid phase can easily be separated. Specifically, after a detection target substance existing in the liquid phase is bound to probe molecules, a conjugate of the detection target substance and probe molecules and unreacted probe molecules are temporarily separated and collected from the liquid phase by using microscopic particles fixing the probe molecules. Thereafter, whether a conjugate of the detection target substance and probe molecules actually exists in a collected mixture of the detection target substance and probe molecules and unreacted probe molecules is determined, and further, the amount of existing conjugate is quantitatively measured.
The advantage that separation from the liquid phase is facilitated by fixing probe molecules on the surface of a solid phase is also achieved by fixing probe molecules on the surface of a solid phase substrate having a macroscopic size. For example, when detecting presence/absence of a specific IgG antibody existing in a specimen sample, an antigen peptide having an epitope sequence to an Ig antibody as a detection target is fixed in a predetermined density on the surface of a solid phase substrate, the specimen sample is brought into contact with the surface of the solid phase substrate, and the IgG antibody as a detection target is fixed on the surface of the solid phase substrate through a reaction with the antigen peptide. Thereafter, the specimen sample is removed to the surface of the solid phase substrate, and an anti IgG antibody given a label for detection is then quantitatively reacted with an Fc region (stationary region) of IgG antibody molecules fixed on the surface of the solid phase substrate. The unreacted “anti IgG antibody given a label” is washed out from the surface of the solid phase substrate, and the anti IgG antibody given a label, which quantitatively reacts with the IgG antibody molecules fixed on the surface of the solid phase substrate, is detected using the label for detection, and quantitatively measured.
In the method of fixing probe molecules on the surface of the solid phase substrate having a macroscopic size, different types of probe molecules can be fixed on regions after sectioning the same substrate surface into a plurality of regions because the solid phase substrate itself is large. Specifically, when sectioning the substrate surface into a plurality of regions, individual sections are arranged in the form of an array or a matrix, and addresses for identifying the sections are then given (lot numbers are given), whereby the form of a probe array with different types of probe molecules fixed on individual sections can be provided. For example, there are forms of: a DNA probe array fixing a plurality of types of DNA nucleic acid probes, which is used for fixation of target nucleic acid molecules including a base sequence portion complementary to a base sequence of a nucleic acid probe, by a hybridization reaction with a nucleic acid probe; a peptide antigen array fixing a plurality of types of antigen peptides having known amino acid sequences, which is used for fixation of target antibody molecules having specificity to antigen peptides, by an antigen/antibody reaction; an antibody array fixing a plurality of types of known specific antibody molecules, which is used for fixation of target antigen molecules using a specific antibody, by an antigen/antibody reaction conversely; and a receptor protein array fixing a plurality of types of known receptor molecules, which is used for fixation of target base molecules to a receptor protein, by the binding of base molecules on the receptor protein.
The probe array is used when a plurality of types of target molecules contained in a specimen sample are fixed at a time using a plurality of types of corresponding probe molecules. Since the solid phase substrate used has a macroscopic size, separation from the liquid phase and subsequent washing operations are extremely easy. When detecting a target substance fixed on the substrate as a conjugate with each probe molecule on the probe array, the fixed position (address) of each probe molecule is determined beforehand, and each target substance is detected in accordance with the address.
The probe array fixing probe molecules on a solid phase substrate surface having a macroscopic size has an advantage that it is easily treated when carrying out separation from the liquid phase and detection of a target substance, and is used in a variety of fields. However, there is a fundamental disadvantage that the reaction yield is relatively low (the apparent reaction rate is low) due to a reaction between probe molecules fixed on a specific region and target molecules contained in a specimen sample on the solid phase substrate surface having a macroscopic size. Specifically, probe molecules are fixed on a limited region on the solid phase substrate surface, but target molecules contained in the specimen sample are uniformly distributed over the entire liquid phase, and therefore target molecules capable of reacting with the probe molecules are limited to those existing within a limited region and on an area adjacent to the solid phase substrate surface. When the target molecules existing within this limited region form a conjugate with fixed probe molecules, the concentration of “free target molecules” existing in the liquid phase within the region abruptly decreases, and resultantly, the reaction rate relatively decreases. As a result, the total number of target molecules forming a conjugate with probe molecules per the total number of fixed probe molecules (reaction yield) is limited. The disadvantage that the reaction yield is relatively low (the apparent reaction rate is low) becomes more noticeable as the ratio of the area of a fixing region of each probe molecule on the solid phase substrate surface decreases. In other words, this disadvantage becomes more noticeable as the number of types of probe molecules constituting the probe array increases.
Ideally, this disadvantage is eliminated by quickly stirring a reaction solution to uniformalize the distribution of concentrations in the liquid phase, but in reality, there are not a few cases where the reaction, is accomplished in a situation in which the reaction solution is gently stirred or almost left stationary when a solid phase substrate surface having a macroscopic size is used. Even if the reaction yield is relatively low (the apparent reaction rate is low), the total number of target molecules forming a conjugate with probe molecules per the total number of fixed probe molecules (reaction yield) reflects the original concentration of target molecules in the specimen solution. However, as the ratio of the area of a fixing region of each probe molecule decreases, the relationship between the total number of target molecules forming a conjugate with probe molecules (reaction yield) and the original concentration of target molecules in the specimen solution becomes harder to show a high linearity. Specifically, when a dense probe array fixing multiple types of probe molecules in the form of a dense matrix on the solid phase substrate surface having a macroscopic size is used, it becomes one of factors of reducing the quantitative accuracy when the concentration of target molecules in the specimen solution is quantitatively measured by quantitatively measurement of target molecules fixed as a conjugate with the probe molecules.
In addition, when constituting a probe array grating-like frame regions for division into sections may be provided on the periphery of fixing regions of probe molecules. Alternatively, even if the grating-like frame regions are not provided, there may be non-fixing regions of, probe molecules on the periphery of fixing regions of probe molecules. There are not a few cases where various target molecules are nonselectively adsorbed because the solid phase substrate surface is exposed on, the grating-like frame regions or the non-fixing regions of probe molecules. The nonselectively adsorbed target molecules raise a signal level of a background at the time of detection, thus causing a systematic error when a difference between a signal level detected in fixing regions of probe molecules and a signal level of a background is determined to be a signal resulting from target molecules forming a conjugate with probe molecules. In addition, they become one of factors of reducing the quantitative accuracy over an area where the concentration of target molecules in the specimen solution is low, since the reaction yield is relatively low (the apparent reaction rate is low).
A spotting method in which probe molecules prepared separately beforehand are coated and fixed on predetermined fixing regions when a probe array is prepared is widely used. In this spotting method, the density of probe molecules fixed per unit surface area has a high reproducibility as in the case where microscopic particles are put in a liquid containing probe molecules prepared separately beforehand and probe molecules are fixed on their surfaces. There is a method in which oligonucleotides synthesized sequentially on a solid phase substrate surface having a macroscopic size in accordance with the base sequence of each DNA probe (oligonucleotide) using a photolithography method is used as fixed probe molecules when DNA probe (oligonucleotide) molecules capable of being synthesized by a solid phase reaction are fixed in the form of an array. For oligonucleotides that are synthesized on the solid phase substrate surface immediately thereafter, it is difficult to observe the base sequence after synthesis, and oligonucleotides lacking bases in part may be present. Otherwise, all (oligo) nucleotides lacking one nucleotide in principle coexist in a certain ratio. Presence of such DNA molecules having base sequences different from desired ones is a factor of relatively reducing the reaction yield in a probe hybridization reaction. In some cases, if DNA molecules having base sequences different from desired ones happen to have base sequences in common with DNA probe molecules fixed on another address, different types of nucleic acid molecules different from original target nucleic acid molecules ate bound on such an address. Thus, presence of the above undesired oligonucleotides becomes one of factors of reducing the quantitative accuracy.
Probe molecule fixing fine particles with probe molecules fixed on the surfaces of microscopic particles are prepared by a method in which microscopic particles are put in a liquid containing probe molecules prepared separately beforehand to fix probe molecules on their surfaces, and therefore a phenomenon in which various target molecules are nonselectively adsorbed to the surfaces of microscopic particles is substantially, inhibited. Probe molecules to be fixed are prepared separately, and then sufficiently purified. As described later, the solid phase sequential synthesis on the surfaces of microscopic particles can avoid the disadvantages of the photolithography method described above, and therefore this method can also be suitably used.
Normally, probe molecule fixing fine particles can be kept in a state of being uniformly dispersed in a liquid phase, and from a microscopic viewpoint, a reaction between the solid phase and the liquid phase occurs with target molecules uniformly distributed over the entire liquid phase, but from a macroscopic viewpoint a uniform reaction can be carried out over the entire liquid phase. Thus, the disadvantage of a relative low reaction yield (low apparent reaction rate) which is noticeable when using a dense probe array fixing multiple types of probe molecules in the form of a dense matrix on the solid phase substrate surface with a macroscopic size is substantially eliminated if the probe molecule fixing fine particles are used. Thus, if probe molecule fixing fine particles are used, most of factors of reducing the quantitative accuracy listed in the probe array can be avoided when the concentration of target molecules in the specimen solution is quantitatively measured by quantitatively measurement of target molecules fixed as a conjugate with probe molecules.
Probe molecule fixing fine particles retains the advantage that normally, they can easily be separated from the liquid phase after completion of a reaction in a state of being uniformly dispersed in the liquid phase. For example, a solid-liquid separation method applying a filtration or centrifugal separation method can be used, and when particles themselves have a magnetic material as a main component, they can be separated from the liquid phase using a magnetic force.
Of course, probe molecule fixing fine particles fix one type of probe molecule on the surface of each microscopic particle, and therefore if multiple types of probe molecules are used, it is necessary to prepare multiple types of corresponding probe molecule fixing fine particles beforehand. These multiple types of probe molecule fixing fine particles have different probe molecules constituting individual probe molecule fixing fine particles, but they cannot easily be discriminated by appearances. In other words, it is necessary that for multiple types of probe molecule fixing fine particles, microscopic particles used be made mutually discriminable and individual probe molecule fixing fine particles be identified. Specifically, it is necessary to give labels for mutual identification to microscopic particles used.
Regarding the method in which labels for mutual identification are given to microscopic particles constituting probe molecule fixing fine particles, some proposals have been made. For example, Japanese Patent Application Laid-Open No. S61-225656 describes a method of identification by a difference in particle diameter of microscopic particles used; Japanese Patent Application Laid-open No. S62-081566 describes a method of performing discrimination using a difference in particle diameter of microscopic particles and fluorescent labels (n types in total) in combination; Japanese Patent Application Laid-Open No. S62-195556 describes a method of performing discrimination by coloring microscopic particles; Japanese Patent Application Laid-Open No. H01-095800 describes a method of performing discrimination by forming microscopic particles with different metal elements; Japanese Patent Application Laid-Open No. H02-299598 describes a method of performing discrimination by staining; Japanese Patent Application Laid-Open No. H07-083927 describes a method of using microscopic particles as inorganic fluorescent materials and performing discrimination by fluorescent wavelengths thereof; U.S. Pat. No. 6,602,671 describes a method of performing discrimination using semiconductor nanocrystals for microscopic particles; U.S. Pat. No. 6,440,667 describes a method of performing discrimination using microscopic particles having different magnetizations, colors and shapes; and U.S. Pat. No. 6,500,622 describes a method of performing discrimination using semiconductor nanofluorescent particles for microscopic particles. They relate to techniques for giving lot numbers (labels) to particles individually.
In these conventional techniques, the number of types of discriminable microscopic particles, specifically the number of types of labels is a dozen or so at most, and the methods are poor in extensibility when the number of types of labels is further increased. Particularly when the number of types of labels is further increased while commonness for the particle diameter, the shape and the main component material of microscopic particles is maintained, the methods are poor in extensibility.
As described above, when for various kinds of biological substances contained in the liquid phase, for example nucleic acid molecules, proteins, sugar chain molecules and the like, the presence/absence of these substances is determined or their contents are measured, a method in which a substance (usually referred to as probe molecules) uniquely bound to an examination object substance (target substance) is made to act on the substance to form a conjugate on a temporary basis and the conjugate is separated from the liquid phase is widely used. Specifically, a method in which probe molecules are fixed on the solid phase surface, whereupon a conjugate is formed with a target substance existing in the liquid phase, and the solid phase and the liquid phase are separated is widely used. After separation from the liquid phase, detection means for adapting the presence/absence of a target substance forming a conjugate with probe molecules and the amount thereof is used.
In recent years a technique of carrying out a PCR (Polymerase Chain Reaction) reaction and a one nucleotide elongation reaction using a terminator nucleotide in conjunction on a solid phase using as a primer a nucleic acid fixed on the solid phase surface has been developed. Japanese Patent Application Laid-Open No. 2001-299346 discloses one method of so called a solid phase PCR in which one set of PCR primers consisting of two types of oligonucleotides is fixed on one matrix of a nucleic acid array to carry out a plurality of PCRs on one solid phase. U.S. Pat. No. 6,004,744 describes one nucleotide elongation using nucleic acid primers fixed on the solid phase.
At this time, as compared to a probe array regularly fixing a plurality of types of probe molecules on a solid phase substrate having a large surface area, probe molecule fixing, fine particles individually fixing probe molecules on the surfaces of fine particles are significantly excellent in reactivity with a target substance in the liquid phase and quantitativeness thereof. However, in the probe array, the type of each probe molecule can easily be identified based on its fixation position (address), but in probe molecule fixing fine particles, it is necessary to give labels to fine particles for use in fixation beforehand and identify the labels to identify the types of probe molecules.